NO843274L - PROCEDURE FOR THE PREPARATION OF AN IMMUNO MODULATOR MEDICINE - Google Patents

Abstract

1. Immunomodulating medicament of biological origin, of which the active principle is formed by the products of lysis or a fraction of these products of bacteria lysed by bacteriophagic route, characterised in that it contains, as active principle, the sterile lysis filtrate of at least two bacteria lysed by homologous bacteriophagic route ; the bacteria chosen being Klebsiella pneumoniae and Escherichia coli.

Description

The present invention relates to immunomodulator drugs of biological origin and their method of manufacture.

Numerous preparations which modulate the immune response via fractions or extracts of lysed microorganisms are known.

One can especially mention the products described in the French patents no. 2 374 042, 2 396 018 and 2 426 470.

These lysates or extracts are generally obtained via physical, chemical and more rarely enzymatic methods. If most of these preparations have a specific immunomodulating activity, brought to light in animals, the therapeutic activities in humans are even more the target of numerous works (Immuno-pharmacologie clinique. Réalités et Perspectives G. RENOUX. Pharmacologie et thérapeutique. R.P. 1982, 32_, 41-42).

It is therefore important to research drugs that "offer both human and veterinary medicine a weapon suitable for use every time it was a question of restoring or strengthening the immune system.

A new method of production has been found

which uses bacteriophage. In vitro, the bacteriophage represents an excellent tool for bacterial lysis; its use in the preparation of immunomodulatory preparations has been effective with various bacterial genera and species. This technique for bacterial lysis by means of bacteriophage relates without limitation to bacterial genera or species. This method has given--few drugs remarkable for their ability to modulate the immune system, produced from one or more bacterial germs or a mixture of these bacterial germs. These drugs in particular have an ability to induce interferons.

These drugs which modulate the immune response whose active principle consists of the lysis products or a fraction of these products of lysed bacteria via bacteriophage, according to the invention, contain as active principle the lysis products from at least two lysed bacteria via homologous bacteriophage.

Among the bacteria, one can mention in a non-limiting way Escherichia coli, Streptococcus faecalis, Klebsiella pneumoniae, Staphylococcus aureus. The preparations obtained by using bacteria from the genera Klebsiella and Escherichia prove to be particularly interesting.

The drug according to the invention can be associated with an antibiotic substance, such as tetracyclines, antivirals such as synthetic nucleosides that interfere with viral nucleic acid synthesis, antifungals, such as buclosamide, or antiparasitics, such as chloroquine.

In another form of execution, the drug according to the invention can be associated with other products and pharmaceutical compositions, such as an anti-tumoral substance (cytostatics), an immuno-suppressive substance, (anti-lymphocytic immunoglobulins) an anti-mitotic substance, such as folic acid derivatives or anti-inflammatory, such as indomethacin.

The medication from the invention can be presented in many forms from percutaneous, transcutaneous, perlingual, topical, enteral, parenteral or respiratory administration. Depending on the case, the drug is manufactured with a suitable binder, such as in the form of an aqueous or alcoholic solution or in suspension in an oil binder, either in lyophilized or aerobic form or ointments.

The dosage used is susceptible to wide variations

in function of the composition of the drug, the prescribed indication, the method of administration, and the age of the treated case. The determination of the treatment doses should be left to the judgment of the doctor or veterinarian, which is common practice in treatments for modulation of the immune response.

As an example, the daily dosage may include the treatment of chronic or recurrent infections in the respiratory tract, in the prevention of postoperative infectious complications, or in the prophylaxis of urinary tract infections, in doses of 1 to 50 ml, depending on the concentration of the immunomodulating preparation.

The drug according to the invention is tolerated in an excellent way, its toxicity is weak as shown by the LD^^ test, which could not have been expected per se considering the stomach capacity of the animal used (mice, rats).

The medicines produced according to the invention are particularly indicated in the treatment of dysregulation of the immune response,

in the prevention or treatment of infectious outbreaks of

bacterial, viral, fungal or parasitic species, such as in rheumatology and in scarring or in the protection of wounds.

The invention also aims to provide a method for the production of a drug of the bacterial lysis type, which corresponds to a frequency of at least two indispensable stages, i.e. a bacteriophage lysis, so the elimination of non-lysed bacteria and bacterial residues by means of a for specialists known method, such as filtration, centrifugation, etc.

According to a method for carrying out the method based on a bacterium, an adapted culture environment is inoculated with a bacterial inoculum, then during the growth phase the bacteria are infected by the addition of their homologous phages, the development of the lysis is followed using a cellular counting apparatus , adjusted in advance to a fixed stage, which corresponds to a number of bacteria per ml in the environment, then you continue with the elimination of non-lysed bacteria and bacterial residues, and you collect the lysis filtrate that makes up the immunomodulating preparation.

The method of the invention can be implemented with the successive use of bacteria of different genera.

In this case, a first inoculation is carried out in a suitable culture environment with a bacterial inoculum of a specific genus during the growth phase, the bacteria are infected by adding their homologous phages, the development of the lysis is followed up to a fixed stage, in the same way as in front, then one inoculates the culture medium with an inoculum of bacteria of another genus, during the growth phase of the bacteria of the other genus, one infects these bacteria by adding their homologous phages, one follows the development of the lysis of the bacteria of this second genus until a fixed stage, finally the non-lysed bacteria and bacterial remains are eliminated using known methods for this type of separation, and the filtrate from the lysing obtained from the two bacteria is collected. According to this method, a drug can be prepared by using successively, for example, bacteria of the genera Klebsiella and Escherichia.

Hereafter, examples are given which illustrate the invention in a non-limiting way.

EXAMPLE 1

Production from a bacterium of

genus Escherichia.

The bacterium used is of the genus Escherichia, species coli, serotype 0119: B 14, registered at the Pasteur Institute in Paris under reference 62.23.

The coliphage belonging to a morphological group C, (nomenclature of the Subcommittee for Taxonomie des Bactériophages of the International Committee for Taxonomy of Viruses of the I.A.M.S.), it has an isometric head, a very short tail, difficult to bring to light, a capsule and a terminal plaque that cannot be detected. In agar culture, the lysis plaque has a diameter of from 1 to 1.5 mm, a round shape, regular contours, an obscured center and confluent clones r<+>.

The cultural environment consists of liter of: beef extract 2.5 g, peptone 4.1 g, sodium chloride 7.0 g, filled with bidistilled water.

The environment is inoculated with a bacterial inoculum which is in an exponential growth phase and the initial density in the culture is 1.10 6 bacteria per ml. Incubation is continued at 37 nC with moderate agitation.

In the middle of the exponential growth phase, ie approximately 60 minutes after inoculation, the bacteria are infected with their homologous phages. The multiplicity of infection is 0.02.

The development of the lysis ring is monitored using a cellular counter, adjusted in advance. When there are less than 1,000 bacteria left per ml, a sterilizing filtration is carried out by passage through a filter membrane with a pore size close to 0.22 nm. The lysis filtrate itself constitutes the immunomodulating preparation.

EXAMPLE 2

Production from a bacterium of

genus Streptococcus.

A bacterium of the genus Streptococcus, species faecalis, variety synogenes, registered at the Pasteur Institute in Paris under reference 53.152 is used.

The phage belongs to a morphological group A^ (nomenclature of the sub-committee for Taxonomie des Bactériophages of the International Committees for Taxonomy of Viruses of the I.A.M.S.), its head is isometric, its tail long, the capsule is visible and the terminal plaque is complex. On agar culture, the lysis plaques are round, less than 0.3 mm, their contours are regular and their center is clear.

There is no clone r + where there is only one.

Per litre, the culture medium has the following composition: 0.3 g beef extract, 1.0 g tryptase, 1.0 g dextrose, 0.5 g sodium chloride, supplemented with ; 2 x distilled water.

The environment was inoculated with a bacterial inoculum that is located

in a stationary phase, and the output density is 1.IO<7> bacteria per ml. It was placed in the dark and at 37°C, with moderate stirring.

At the beginning of the exponential growth phase, ie approximately 40 minutes after inoculation, the bacteria were infected with their homologous phages. The multiplicity of infection is 0.1.

The development of the lysis was followed using a cellular counter. When there are 10 bacteria left per ml, the culture is sterile centrifuged at 5000 G to eliminate the lysogenic bacteria and the non-lysed bacterial organisms. The super-natant constitutes the immunomodulating preparation.

EXAMPLE 3

Production with the successive use of bacteria

of the genera Klebsiella and Escherichia.

The bacterium of the genus Klebsiella, species pneumoniae, is registered by the Pasteur Institute in Paris under reference 58.5.

The characteristics of the bacterium of strain Escherichia are those described in Example 1.

The phage, homologous to Klebsiella, belongs to a morphological group C-^ (nomenclature of the sub-committee for Taxonomie des Bactériophages of the International Committee for Taxonomy of Viruses of the I.A.M.S.), it has an icosahedral head, a short tail, difficult to bring to light, the capsule and the terminal plaque_£r it is not possible to detect. On agar culture, the lysis plaques measure 0.5-3 mm and are of a very irregular shape, with blurred contours and a clear centre. The clones<r+>are rare.

The characteristics of the coli phage are described in example 1.

Per litre, the culture medium has the following composition: 3 g beef extract, 5 g peptone, 7 g sodium chloride, filled with 2 x distilled water.

The environment was inoculated with an inoculum of Klebsiella pneumoniae which is in a stationary growth phase. The output density is 1.10<7> bacteria per ml. The culture is placed at 37°C with moderate agitation.

At the end of the latency phase, i.e. approximately 60 minutes after inoculation, the bacteria are infected with their homologous phages, the multiplicity of infection being 0.03.

The development of lysis was followed using a cellular

counting device. When there are less than 10 bacteria left per ml, the environment is re-inoculated with an inoculum of Escherichia which is in a stationary weight phase. The initial density of this new culture is 5.10 7 Escherichia per ml.

At the end of the latent phase, i.e. under these experimental conditions, 20 minutes after the second inoculation, the bacteria are infected with coliphages. The multiplicity of infection is 0.1.

The development of lysis was followed using a cellular counting apparatus. When there are less than 10 3 Escherichia per ml, the culture is stopped by passage through a sterilizing membrane filter of 0.22 nm. The lysis filtrate obtained from the two bacteria, Klebsiella pneumoniae and Escherichia coli, constitutes the immunomodulating preparation.

EXAMPLE 4

Production by using a bacterium of the genus Staphylococcus.

The bacterium of the genus Staphylococcus, species aureus, serotype 1, is registered by the Pasteur Institute in Paris under reference 65.6.

The homologous phage of Staphylococcus belongs to a morphological group (nomenclature of the sub-committee for Taxonomie des Bactériophages of the International Committee for Taxonomy of Viruses of I.A.M.S.); it has an octahedral head and a tail covered by a capsule and a complex terminal plaque. On agar culture, the lysis plaques have a diameter of 1-2 mm, they are round and regular

re, their center is clear, there is no clone

The bacteria are dispersed in a fixed environment that contains per liter: 10.0 g 3 Difco peptone proteose, 1.2 g dextrose, 6.0 g soluble starch, 3.0 g sodium chloride, 2.0 g sodium diphosphate, 13.0 g bacto-gelatin, 6.0 g bacto-agar , purified water q.s.

After 24 hours, the germs that form a dense bacterial blanket are harvested sterilely, counted and resuspended in an environment that

contains per litre: 2.5 g beef extract, 4.1 g peptone,

7.0 sodium chloride, 2.x distilled water q.s., to obtain a density of 5.10 bacteria per ml.

At the same time, the homologous subjects are added to the environment. The multiplicity of infection is 0.01.

The development of the lysis ring is followed using a cellular counting apparatus. When there are less than 10 2 bacteria left per ml, a filtration over a membrane with a pore size close to 0.22 pm is carried out. The lysis filtrate itself constitutes the immunomodulating preparation.

Pharmacological studies:

The effects on animals of preparations according to the invention are illustrated with preparations corresponding to a lysis filtrate of Escherichia coli obtained according to the method described in example 1. The filtrate was lyophilized, then redissolved in distilled water. The concentration factor of this new solution is expressed in relation to the preparation before lyophilization.

Protective effect against an acute bacterial infection:

This test, which brings to light a total stimulation of the immune system, has been carried out with infectious germs different from those included in the preparation. This is to prove that the preparation is not effective via a possible vaccinating ability or via a direct bacteriophagic phenomenon, but really via a stimulation of the immune system.

Two germs have been tested, Klebsiella pneumoniae, a bacterium with intracellular development and of Staphylococcus aureus,

a bacterium with extracellular development.

The infecting bacteria were administered to groups of 10 "Swiss" female mice, with an average weight of 18 g, on day D^, via the intraperitoneal route in a volume of 0.5 ml.

The immunomodulating preparation was injected the day before the infection. The preparation was administered via the intraperitoneal or intravenous route, in order to remove any positive result caused by a direct interference of the preparation with the infecting germs at the injection site.

To determine the virulence of the infecting strain, which can vary within considerable limits from one experiment to another, its lethal dose 50 was determined for each of the experiments. The protective activity of the preparation against an acute bacterial infection is shown in Table I below.

Stimulation of the phagocytic action of macrophages

Halpern's test (C. Stiffel, 1958, J. Phys. 58, 911-949) consists in injecting mice via the intravenous route with colloidal carbon particles which are phagocytosed by the reticulo-endothelial cells, mainly in the liver and spleen. Their clearance rate is representative of the phagocytic activity. It is measured by determining quantitatively via photometry as a function of time, the particles that have remained free in the blood. LPS, a lipo-polysaccharide endotoxin extract of enterobacteria and strong activator of macrophages, was used as a reference substance.

The test was carried out as follows:

0.6 ml of a filtrate of Escherichia coli or a solution of LPS (lipopolysaccharides) at 0.625 µg/ml is injected into groups of 5 female "Swiss" mice, with an average weight of 24 g, via the intraperitoneal route.

24 hours later, 0.12 ml of a suspension of colloidal carbon dosed to 32 mg/ml carbon in 4% gelatin is injected to

mice via the intravenous route.

A blood sample of 20 µl was taken from the tetro-orbital sinus every two minutes for 12 minutes. This blood sample is immediately dissolved in 4 ml of 1% Na2C0^ The solution is therefore photometric.

The reading of the extinction is done at 620 nm. The concentration of colloidal carbon in the blood at time t

in the experiment is obtained by referring to a standard location chosen in advance. The phagocytic index is calculated according to the following formula:

C2 and represent respectively the blood concentration of colloidal carbon in the second of the first blood sample, T2 and represent the time for these blood samples.

The results recorded in Table II show that the effect of

the peritoneal macrophages in mice are stimulated after an intraperitoneal administration of the preparation.

^ Inducing effect of serum interferon (serial IFN)

IFN is a glycoprotein with a molecular weight between 10,000 and 20,000 daltons. It has numerous immunomodulating effects,

in particular, it stimulates the action of NK (Natural Killer) cells. Its mode of action is under study. Its participation in the antiviral defense has indeed been demonstrated.

Two hours after an administration of the preparation to 8 "Swiss" female mice of 25 grams, 0.3 ml of blood is collected from each of them in a tube without heparin. Serum IFN is measured quantitatively in a classical way by measuring the cytopathic effect of the virus VSV (Vesicular Stomatitis Virus) infecting murine L. cells cultured for 24 hours in the presence of the serum to be tested.

(E.A. Havell and J. Vilcék, Antimicrob. Agents Chemotherapy, 2, 476-484, 1972). The protective effect of the induced IFN is compared with that of the murine IFN, the international reference being from the National Institute of Health, Bethesda, USA.

The preparation induces serum IFN in mice 2 hours after its intraperitoneal administration, intravenously or orally, as indicated in Table III.

The effect in humans has been tested with a preparation according to example 3, administered first to volunteers, then to patients.

A double blind study carried out with 15 volunteer patients has made it possible to bring to light that the oral administration of the preparation after 10 days of treatment led to a significant increase in the immune response during cellular transmission.

Another study carried out on 25 patients has proven that a single oral administration of different doses of the preparation after 24 hours allowed to induce a serum lymphokine of the MIF-LIF type. This induction is dose dependent.

Several studies have allowed to bring to light the stimulation of the production of IFN by human lymphocytes and monocytes obtained by cytopheresis and cultured in teats.

IFN induced by the preparation has been able to be characterized by using antisera anti-IFN a, j3 or y. Its production depends on the applied dose.

As examples, some clinical works are referred to below.

Prevention and treatment of chronic or recurrent infections in the respiratory tract.

Two groups of 25 patients attacked by chronic or recurrent bacterial infections at the broncho-pulmonary level were treated orally respectively, one with 15 ml of the above-mentioned preparation, 20 days a month for 3 months, the other with the classic antibiotic treatments. The reduction in the number of acute infectious cases during the six months that the trial lasted is significantly greater in the group treated with the preparation than in the group treated classically with antibiotics.

Prevention of post-operative infections:

An oral administration of 20 ml of the preparation has significantly reduced the infectious post-operative complications in 106 patients compared to 72 other patients treated in the classical way.

Prevention and treatment of urinary tract infections: Four researchers have clinically studied the preparation above, in a population of patients affected by chronic or recurrent urinary tract infections. An oral administration of 15 ml per day, 20 days a month during 3 months has significantly allowed to reduce the frequency and duration of infectious cases and also the consumption of anti-infectives during the six months of observation compared to the six months preceding the treatment.

In all trials, the tolerance of the preparation has been excellently assessed by the doctor and the patient (volunteer or sick).

Claims (9)

1. Immunomodulating drug of biological origin, whose active principle consists of lysis products or a fraction of these products of lysed bacteria via bacteriophage, characterized in that it contains as active principle the lysis products of at least two lysed bacteria via homologous bacteriophage. 2. Immunomodulating drug according to claim 1, characterized in that it contains as active principle the lysis products via homologous bacteriophage from the bacteria of the genera Klebsiella and Escherichia. 3. Immunomodulating drug according to claim 2, characterized in that the selected bacteria are Klebsiella pneumoniae and Escherichia coli. 4. Medicine according to claim 1, characterized in that it also contains an antibiotic, anti-viral, anti-fungal or anti-parasitic substance. 5. Medicine according to claim 1, characterized in that it also contains an anti-tumoral or immuno-suppressive substance. 6. Medicine according to claim 1, characterized in that it also contains an anti-mitotic or anti-inflammatory substance. 7. Medicine according to claim 1, characterized in that the active principle is associated with a binder adapted for percutaneous, transcutaneous, perlingual, topical, enteral, parenteral or airway administration. 8. Method for producing the drug according to claim 1, characterized by a first inoculation in a suitable culture environment with an inoculum of bacteria of a specific genus, during the growth phase the bacteria are infected by adding their homologous phages, follows the development of lysis to a fixed stage, so a second inoculation is carried out in the culture medium with an inoculum of bacteria of another genus, during the growth phase of the bacteria of the other genus these bacteria are infected by adding their homologous phages, the development of lysis of these bacteria of the other is followed strain to a fixed stage, finally the non-lysed bacteria are eliminated, the lysis filtrate from the two bacteria is collected. 9. Application of the drugs in curative or preventive therapy of bacterial, viral, fungal or parasitic infections; in antimitotic or anti-inflammatory therapy; as an adjuvant in immuno-suppressive or anti-cancerous therapy.